The finite proliferative potential of normal human cells leads to replicative cellular senescence, which is a critical barrier in tumour progression in vivo. We have shown that human p53 isoforms (delta 133p53 and p53beta) function in an endogenous regulatory mechanism for p53-mediated replicative senescence. Induced p53beta and diminished delta133p53 were associated with replicative senescence, but not oncogene-induced senescence, in normal human fibroblasts. The replicatively senescent fibroblasts also expressed increased levels of miR-34a, a p53-induced microRNA, the antisense inhibition of which delayed the onset of replicative senescence. The short interfering RNA (siRNA)- mediated knockdown of endogenous delta133p53 induced cellular senescence, which was attributed to the regulation of p21waf1 and other p53 transcriptional target genes. In overexpression experiments, whereas p53beta cooperated with full-length p53 to accelarate cellular senescence, delta133p53 repressed miR-34a expression and extended the cellular replicative lifespan, providing a functional connection of this microRNA to the p53 isoform-mediated regulation of senescence. The senescence-associated signature of p53 isoform expression (that is, elevated p53beta and reduced delta133p53) was observed in vivo in colon adenomas with senescent phenotypes. The increased delta13p53 and decreased p53beta isoform expression found in colon carcinoma may signal an escape from the senescence barrier during the progression from adenoma to carcinoma. Senescence is a tumor suppression mechanism that is induced by several stimuli, including oncogenic signaling and telomere shortening, and controlled by the p53/p21(WAF1) signaling pathway. Recently, a critical role for secreted factors has emerged, suggesting that extracellular signals are necessary for the onset and maintenance of senescence. Conversely, factors secreted by senescent cells may promote tumor growth. By using expression profiling techniques, we searched for secreted factors that were overexpressed in fibroblasts undergoing replicative senescence. We identified WNT16B, a member of the WNT family of secreted proteins. We found that WNT16B is overexpressed in cells undergoing stress-induced premature senescence and oncogene-induced senescence in both the MRC5 cell line and the in vivo murine model of K-Ras(V12)-induced senescence. By small interfering RNA experiments, we observed that both p53 and WNT16B are necessary for the onset of replicative senescence. WNT16B expression is required for the full transcriptional activation of p21(WAF1). Moreover, WNT16B regulates activation of the phosphoinositide 3-kinase (PI3K)/AKT pathway. Overall, we identified WNT16B as a new marker of senescence that regulates p53 activity and the PI3K/AKT pathway and is necessary for the onset of replicative senescence.